There is a balance between factors promoting renal salt and water excretion (e.g., nitric oxide (NO)) and those favoring retention (e.g., angiotensin II). Inappropriate salt retention may lead to hypertension. This project focuses on one factor favoring salt and water excretion, NO. An important aspect of NO's antihypertensive action is regulation of renal function. The thick ascending limb absorbs 20-30% of the filtered NaCl load, which is essential for proper Na balance. We have reported that NO produced by NO synthase 3 (NOS 3; also known as endothelial NO synthase) in this segment acts as an autacoid to inhibit transport. In endothelial cells, flow augments NOS 3 activity via mechano-sensitive channels. Our preliminary data indicate that luminal flow is an important activator of NOS 3 in the thick ascending limb and that activation requires translocation of the enzyme to the luminal membrane. However, it is not known how luminal flow regulates NOS 3 activity in the thick ascending limb or how flow-induced NO affects NaCl reabsorption. We hypothesize that increasing luminal flow in the thick ascending limb stimulates NO production by NOS 3 via activation of mechanosensitive channels, release of ATP and activation of Akt. The NO thus produced blunts NaCl reabsorption. This hypothesis will be tested in 4 aims. Aim I will test whether increasing luminal flow causes translocation and activation of NOS 3, stimulating NO production by the thick ascending limb due to an increase in shear stress rather than pressure, stretch or enhanced delivery of Na, K, HCO3 or Cl. Aim II will study whether increasing luminal flow enhances NOS 3 activity by activating mechano-sensitive channels and causing local increases in intracellular Ca. Aim III will test whether luminal flow induces the release of ATP, which binds to purinergic type 2 (P2) receptors and activates the phosphatidylinositol 3 kinase (PI3 kinase)/Akt signaling cascade. Aim IV will investigate whether flow-induced increases in NO production inhibit Na reabsorption by the thick ascending limb via reductions in cAMP. These will be the first studies to define the mechanisms by which flow activates NOS 3 and the effects on NaCl reabsorption by the thick ascending limb. Definition of such pathways will provide new insights into the role of NO in the regulation of urinary volume and salt excretion and may lead to new therapeutic targets for the treatment of hypertension. [unreadable] [unreadable] [unreadable]